Plasmid transformation and curing of nalidixic acid gene in Staphylococcus aureus isolated from buffaloes mastitis and workerʼs hands

The present study was designed to detect resistant site of nalidixic acid through transformation and plasmid curing of S.aureus strains isolated from buffalo milk with subclinical mastitis and workers’ hands. A total of 37 S. aureus isolatesincluding 17 isolates recovered from buffalo milk infected with subclinical mastitis, in addition to 20 isolates recovered fromworkers’ hands. All 37 isolates were investigated by detection of the 23S rRNA gene and various other species specific genesincluding coa, nuc and clfA. The antibiotic resistance of S. aureus isolates was performed by the discs diffusion method using19 antibiotics. Plasmid transformation method was carried out by transferring the plasmid isolated from S. aureus intocompetent Escherichia coli HB 101 in order to detection the resistant site of nalidixic acid. Plasmid curing was accomplishedby preparing different concentrations of nalidixic acid (100, 150, 200, 250 and 300 μg/ml) and cultured transformed E.coli onLB agar supported with each of the aforementioned concentrations. The molecular results showed that six isolates (fiveisolates from milk samples and one from workers’ hands) were identified as S. aureus by coa, nuc, and clfA species specificprimers. The six S. aureus isolates were found to be resistant to at least 5 antibiotics which included the nalidixic acid. Theresults of plasmid transformation revealed that E. coli was able to grow on LB agar supported with 100μg/ml, 150 μg/ml, 200μg/ml and 250 μg/ml of nalidixic acid and failed to grow on 300 μg/ml concentration.